The present invention is in the field of secreted proteins that are related to the lactate dehydrogenase secreted protein subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Secreted Proteins
Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. The present invention advances the state of the art by providing many novel human secreted proteins.
Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis.
Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a xe2x80x98signal sequence trapxe2x80x99 technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer.
Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth. Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al., Mutat Res 1991 March-November; 256(2-6):127-38).
The secreted form of amyloid beta/A4 protein precursor (APP) functions as a growth and/or differentiation factor. The secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms. (Roch et al., Ann N Y Acad Sci Sep. 24, 1993; 695:149-57). Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. The prominent effects of secreted APPs on synaptogenesis and neuronal survival suggest that secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer""s disease (Mattson et al., Perspect Dev Neurobiol 1998; 5(4):337-52).
Breast cancer cells secrete a 52K estrogen-regulated protein (see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secreted protein is therefore useful in breast cancer diagnosis.
Two secreted proteins released by platelets, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators of platelet involvement in hemostasis and thrombosis and assays that measure these secreted proteins are useful for studying the pathogenesis and course of thromboembolic disorders (Kaplan, Adv Exp Med Biol 1978;102:105-19).
Vascular endothelial growth factor (VEGF) is another example of a naturally secreted protein. VEGF binds to cell-surface heparan sulfates, is generated by hypoxic endothelial cells, reduces apoptosis, and binds to high-affinity receptors that are up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996 September;1(3):225-32).
Many critical components of the immune system are secreted proteins, such as antibodies, and many important functions of the immune system are dependent upon the action of secreted proteins. For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secreted IgE proteins.
For a further review of secreted proteins, see Nilsen-Hamilton et al., Cell Biol Int Rep 1982 September;6(9):815-36.
Lactate Dehydrogenase
Lactate dehydrogenase (LDH), sometimes referred to as lactic dehydrogenase, is the most clinically important dehydrogenase occurring in human serum. LDH is clinically important because serum level of certain isozymes reflect pathological conditions in particular tissues. Consequently, LDH is most often measured to evaluate the presence of tissue damage. LDH serves as an indicator suggestive of disturbances of the cellular integrity induced by pathological conditions. Since LDH is an enzyme present in essentially all major organ systems, serum LDH activity is abnormal in a large number of disorders.
LDH is found in the cytoplasm of cells and catalyzes the following reversible reaction for the interconversion of pyruvate and lactate: Pyruvate+NADH←xe2x86x92Lactate+NAD
This bidirectional reaction can be monitored spectrophotometrically by measuring either the increase in NADH at 340 nm produced in the lactate-to-pyruvate reaction, or by measuring the decrease in NADH at 340 nm produced in the pyruvate-to-lactate reaction.
Mammalian LDH exists as five tetrameric isozymes composed of combinations of two different polypeptide subunits with a molecular weight of 136,700xc2x12,100 daltons per tetramer. These tetrameric isozymes each differ in catalytic, physical, and immunological properties. Cahn et al., (1962) designated the two polypeptide subunits as H (heart) and M (muscle), which combine to form two pure types of isozymes, H4 and M4, and three hybrids, H3M, H2M2 and HM3. Type H4 is the most negatively charged at pH 7 and appears nearest the anode upon zone electrophoresis. Subunit H predominates in heart muscle and facilitates the aerobic oxidation of pyruvate. The M subunit predominates in skeletal muscle and liver and is predominantly involved with anaerobic metabolism and pyruvate reduction.
Iodide inhibits LDH; p-mercuribenzoate also inhibits LDH, but at a slower rate. LDH is activated by a number of organic compounds that stabilize the enzyme, such as dimethyl sulfoxide, ethanol, and methanol. Diethystilbestrol and several of its derivatives also stabilize the enzyme.
Normal total LDH levels are approximately 105 to 333 IU/L (international units per liter). Higher than normal LDH levels may be indicative of such conditions as cerebrovascular accident (e.g., CVA, stroke), myocardial infarction, hemolytic anemia (as well as other forms of anemia), hypotension, infectious mononucleosis, intestinal ischemia and infarction, liver disease (e.g., hepatitis), muscle injury, muscular dystrophy, neoplastic conditions, pancreatitis, and pulmonary infarction. If the total LDH level is elevated, specific serum LDH isoenzymes may be measured to increase the accuracy of diagnosis.
The most frequent use of LDH isoenzyme analysis is for the diagnosis of myocardial infarction and other myocardial diseases. Serum LDH levels are elevated in various myocardial diseases and a certain pattern of LDH isoenzyme elevation occurs in myocardial disease. In the absence of hemolysis, an LDH-1/LDH-2 ratio greater than 1 (referred to as a xe2x80x9cflippedxe2x80x9d ratio) is usually indicative of acute myocardial infarction (AMI). This flipped ratio is observed in about 80% of AMI patients. The normal ratio rarely exceeds 0.80 in the absence of AMI. LDH activity begins to rise 8-12 hrs after the onset of chest pain, peaking at 24-48 hrs, and elevated levels may persist for 1 week or more.
LDH activity and its isoenzyme pattern are also useful indicators of pathological conditions in the lungs, such as cell damage or inflammation. Measurement of LDH activity levels and its isoenzyme pattern in pleural effusion and in bronchoalveolar lavage fluid may provide substantial information about lung and pulmonary endothelial cell injury.
LDH isoenzymes are also useful for evaluating other physiological conditions and disorders such as muscle trauma, liver damage, and a variety of malignancies. In these cases, variations of isoenzyme distribution are useful diagnostic indicators. Serum LDH levels are also useful for diagnosing ovarian dysgerminoma and testicular germ cell tumors. Furthermore, LDH is also useful for establishing the survival duration and rate in Hodgkin""s disease and non-Hodgkin""s lymphoma, and in the follow-up of ovarian dysgerminoma. Additionally, LDH is also useful for a variety of NAD, NADH, NADP and NADPH related studies.
For a further review of lactate dehydrogenases, see: Li, Prog Clin Biol Res 1990;344:75-99; Markert et al., Cell Biochem Funct 1984 July;2(3):131-4; Huijgen et al., Eur J Clin Chem Clin Biochem 1997 August;35(8):569-79; Drent et al., Eur Respir J 1996 August;9(8):1736-42; Vanderlinde, Ann Clin Lab Sci 1985 January-February; 15(1):13-31; and Wolf, Clin Lab Med 1989 December;9(4):655-65.
Secreted proteins, particularly members of the lactate dehydrogenase secreted protein subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of secreted proteins. The present invention advances the state of the art by providing previously unidentified human secreted proteins that have homology to members of the lactate dehydrogenase secreted protein subfamily.
The present invention is based in part on the identification of amino acid sequences of human secreted peptides and proteins that are related to the lactate dehydrogenase secreted protein subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate secreted protein activity in cells and tissues that express the secreted protein.